Simultaneous quantitation of the 5′-triphosphate metabolites of zidovudine, lamivudine, and stavudine in peripheral mononuclear blood cells of HIV infected patients by high-performance liquid chromatography tandem mass spectrometry

• Published in: Journal of the American Society for Mass Spectrometry Vol. 11; no. 12; pp. 1134 - 1143
• Main Authors: Moore, Jeff D, Valette, Gilles, Darque, Albert, Zhou, Xiao-Jian, Sommadossi, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 01.12.2000; Elsevier Science; Springer Nature B.V
• Subjects: Alkaline phosphatase; Analysis; Anti-HIV Agents - blood; Antiretroviral drugs; Biological and medical sciences; Blood cells; Calibration; Chromatography; Chromatography, High Pressure Liquid; Cytidine Triphosphate - analogs & derivatives; Cytidine Triphosphate - blood; Dideoxynucleotides; Drug Therapy, Combination; Extraction; General pharmacology; High performance liquid chromatography; HIV Infections - blood; Human immunodeficiency virus; Humans; Lamivudine - analogs & derivatives; Lamivudine - blood; Mass Spectrometry; Medical sciences; Metabolites; Monocytes - chemistry; Monocytes - metabolism; Nucleosides; Nucleotides; Patients; Pharmacology. Drug treatments; Quadrupoles; Reference Standards; Scientific imaging; Sensitivity and Specificity; Spectroscopy; stavudine; Stavudine - blood; Stavudine - pharmacokinetics; Thymine Nucleotides - blood; Zidovudine - analogs & derivatives; Zidovudine - blood; Biological fluid; Metabolite; Coupled method; Retroviridae; HPLC chromatography; Lamivudine; Stavudine; Lentivirus; Quadrupole spectrometer; Blood plasma; Virus; Mononuclear cell; Dideoxynucleoside; Mass spectrometry MS/MS; Simultaneous measurement; Antiviral; Human immunodeficiency virus; Pyrimidine nucleoside; Zidovudine; Quantitative analysis
• DOI: 10.1016/S1044-0305(00)00178-1
• ISSN: 1044-0305

A high-performance liquid chromatography (HPLC) method utilizing triple quadrupole mass spectrometry (MS) detection was developed and validated for the simultaneous measurement of the intracellular nucleoside 5′-triphosphate anabolites of zidovudine (ZDV-TP), lamivudine (3TC-TP), and stavudine (d4T-TP). These compounds were extracted from patient peripheral blood mononuclear cells (PBMCs) which are the sites of HIV replication and drug action. Ion-exchange solid phase extraction (SPE) followed by enzymatic digestion with alkaline phosphatase was utilized to yield the measurable nucleoside forms of the nucleotides. Reversed phase C-18 SPE with addition of a nucleoside internal standard, 3′-azido-2′,3′-dideoxyuridine (AzdU) allowed for the indirect measurement of the original 5′-triphosphate concentration by HPLC/MS/MS. Quantitation was performed from calibration curves generated from authentic 5′-triphosphate standards spiked in PBMCs from healthy volunteers. Analytical range for the three 5′-triphosphates was equivalent to 50–45,000 pg. Mean interassay accuracies for 3TC-TP, d4T-TP, and ZDV-TP ( n > 90) were 99.4%, 100.1%, and 108.0%, respectively. Mean interassay precisions (%C.V.) for 3TC-TP, d4T-TP, and ZDV-TP ( n > 90) were 8.8%, 10.4%, and 8.2%, respectively. Recovery of the extraction method was 79.2%, 83.1%, and 98.3% for 3TC-TP, d4T-TP, and ZDV-TP, respectively. This method can be utilized to measure the intracellular 5′-triphosphate levels in HIV infected patients receiving antiretroviral therapy containing the nucleoside reverse transcriptase inhibitors 3TC, d4T, or ZDV.